1. Field of the Invention
The present invention provides novel conjugates for use in targeting a selected agent, and particularly, asteroid, to vascular endothelial cells. These conjugates comprise two components preferably linked by a selectively-hydrolyzable bond, such as an acid-labile bond. Firstly, a polyanionic polymer which directs the conjugate to vascular endothelial cells, and secondly, a selected agent, such as asteroid, which exerts its action following cellular release. In certain aspects, the invention provides novel conjugates that function as targeted angiogenesis inhibitors that are proposed for use in the treatment of pathological conditions such as cancer, arthritis, and diabetic blindness. Preferred inhibitors are those in which the polyanionic polymer is a polysulfated polymer such as a heparin derivative, conjugated to asteroid with anti-angiogenic activity, such as cortisol, or derivatives and variants thereof.
2. Description of the Related Art
The control of endothelial cell proliferation is a vital part of normal homeostatic mechanisms. A disturbance in this process can result in, for example, excessive or inappropriate endothelial cell proliferation or activation which is often associated with disease processes. For example, the proliferation of vascular endothelial cells is vital to angiogenesis, the formation of new blood vessels, which in turn, is associated with many disabling or life-threatening disorders. These include cancer (Algire et al., 1945; Tannock, 1968; Folkman, 1972) and other pathological conditions such as diabetic retinopathy, atherosclerosis, rheumatoid arthritis, synovitis, psoriasis, dermatitis, endometriosis, encephalitis and tonsillitis (Brown & Weiss, 1988, Waltman et al., 1978; Gartner & Henkind, 1978; Moses & Langer, 1991). It is known that angiogenesis rarely occurs in healthy adult humans except during wound healing and during phases of the female reproductive cycle (Hobson & Denekamp, 1984).
In solid tumors, vascular endothelial cells divide about 35 times more rapidly than those in normal tissues, except the uterine epithelium (Denenkamp & Hobson, 1982). Such inappropriate proliferation is necessary for tumor growth and metastasis (Folkman, 1986). Vascular endothelial cell growth and division is also important in chronic inflammatory diseases such as rheumatoid arthritis, psoriasis and synovitis, where these cells proliferate in response to growth factors released within the inflammatory site (Brown & Weiss, 1988). In atherosclerosis, formation of the atherosclerotic plaque is triggered by a monoclonal expansion of endothelial cells in blood vessels (Alpern-Elran et al., 1989). Furthermore, in diabetic retinopathy, blindness is thought to be caused by basement membrane changes in the eye, which stimulate uncontrolled angiogenesis and consumption of the retina (West & Kumar, 1988).
Endothelial cells are also involved in graft rejection. In allograft rejection episodes, endothelial cells express proadhesive determinants that direct leukocyte traffic to the site of the graft. It is believed that the induction of leukocyte adhesion molecules on the endothelial cells in the graft may be induced by locally-released cytokines, as is known to occur in an inflammatory lesion.
As endothelial cells are involved in a wide variety of processes, it has been reasoned that drugs targeted to such cells may be of wide-ranging use clinically. Perhaps most importantly, angiogenesis inhibitors could potentially be of use in the treatment of those diseases, mentioned above, whose pathogenesis is influenced or maintained by the proliferation of vascular endothelial cells. Angiogenesis inhibitors would be particularly advantageous in the treatment of cancer, in which one of the current major problems is the emergence of drug-resistant malignant cells.
Within the last decade, several inhibitors of angiogenesis have been identified (Langer et al., 1976; Taylor & Folkman, 1982; Sharpe et al., 1990, Good et al., 1990, Ingber et al., 1990). For example, anti-angiogenic therapy, in the form of oral or subcutaneous administration of heparin and the steroid cortisone, has been reported to have an anti-tumor effect in mice bearing established tumors of various types (Folkman et al., 1983). It was believed that the heparin, or heparin metabolites, acted together with the cortisone to inhibit angiogenesis and tumor growth. Unfortunately, the magnitude of the anti-tumor effect varied with the batch of heparin used, and later studies from several laboratories have subsequently reported no (Ziche et al., 1985; Penhaligon & Campejohn, 1985), or only modest (Sakamoto et al., 1986; Benrezzak, 1989), anti-tumor effects.
The mechanism underlying the additive anti-angiogenic activity of heparin and cortisone has not been positively identified, although it may be related to the fact that the mixture increases the rate of dissolution of the basement membrane beneath newly formed capillaries (Ingber et al., 1986). Structure-activity studies have shown that steroids lacking glucocorticoid and mineralocorticoid activity can act in conjunction with heparin to inhibit angiogenesis. These studies have led to the description of a new biological activity of steroids, called the angiostatic activity (Crum et al., 1985).
More recently, the synthetic heparin substitute, .beta.-cyclodextrin tetradecasulfate, was reported to augment the anti-angiogenic effects of angiostatic steroids on corneal neovascularization in rabbits when applied locally or topically (Folkman et al., 1989). It was suggested that the .beta.-cyclodextrin tetradecasulfate might act by forming a non-covalent complex with the steroid and promote its binding to the surface of endothelial cells.
Other potential uses of modulating endothelial cell activity include the treatment of inflammatory responses or the stimulation of growth and proliferation. In the latter instance, this would prove useful in stimulating blood vessel growth, for example, in wound repair after accidental injury or surgery, or in promoting the healing of gastrointestinal lesions such as ulcers. Again, various steroids have potential for use in this area.
The clinical use of many agents, including steroids, whether designed to be inhibitory or stimulatory, is often limited by their side effects and toxicities. For example, steroids are known to have negative effects on bone and lymphoid tissues, which can reduce their overall dosages and their therapeutic effectiveness. In particular, steroid treatment is known to be associated with osteoporosis and immunosuppression. Naturally, these are serious drawbacks which currently limit the use of steroids in the treatment of human disorders. Most toxicity can generally be attributed to a lack of selective action, i.e. it is due to the tendency of the drug to exert its effects on cells indiscriminately.
Accordingly, there is currently a substantial need for an improved means of treating diseases or processes involving endothelial cells, and particularly, processes involving inappropriate endothelial cell growth or proliferation. Furthermore, there is a particular need for this means of treatment to have increased selectivity and specificity, allowing the advantageous use of various agents whilst limiting any toxicity of the agents on tissues other than the endothelial cell tissue.